A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

Abstract

All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.

Keywords: Genetic engineering; Molecular mechanism of gene regulation; Molecular microbiology.

Graphical abstract

Figure 1

Workflow of synthetic sRNA cloning, library generation and characterization (A) Synthetic sRNAs for post-transcriptional control can have a short length and a simple structure consisting of the 5′ seed region and the sRNA scaffold. The seed region can be modified for specific targeting of mRNAs. (B) Based on the simple structure, sRNAs can be modularized and constructed by Golden Gate cloning. The transcription unit of the constructed synthetic sRNAs consists of the promoter (in this case P_LlacO-1), a seed region and the sRNA scaffold (in this case RybB). The three parts are combined into the appropriate acceptor plasmid for subsequent experiments. (C) Concept of oligonucleotide pool-based libraries. The pooled oligonucleotide sequences contain 5′ and 3′ universal sequences providing the required Type IIS recognition and cut sites (the Type IIS recognition site is visualized by the gray box with arrows for its direction and the cut site is highlighted by the indicated nucleotide sequence of 3 nt in length). The universal sequences serve as primer binding sites (indicated by the outermost arrows) to convert the single-stranded oligonucleotide pool into a double-stranded DNA library that can subsequently be used for cloning approaches. Different colors of oligo pool sequences indicate different seed region lengths in the library. (D) Schematic workflow for construction and selection of synthetic sRNAs. The workflow can be completed within <10 days depending on the chosen sequence validation method to obtain characterized synthetic sRNAs for their application.

Figure 2

Initial screening and detailed characterization of the RybB SRL-library targeting acrA mRNA (A) Screening of RybB SRL-library. 380 candidates and four respective controls were spotted from fresh overnight cultures into 3 × 3 grids on LB-agar plates with and without oxacillin. Comparing the reference and the testing condition indicates multiple candidates render cells susceptible toward oxacillin. Comparing the established pP_L-RybB-s8 construct with the library indicates sRNA constructs with a superior regulation. Violet box indicates the position of indicated controls. (B) Liquid growth analysis of the SRL-library indicates regulation for the majority of synthetic sRNAs by comparing the area under the curve (AUC) in the presence and absence of oxacillin. According to the assay, at least 14 nt are needed for regulation of acrA mRNA. Further, the SRL does not correlate with its functionality despite possessing lower binding energies. Binding energies were calculated by IntaRNA (for display reasons the values are multiplied by −1). Wild type (wt) and ΔacrA with an empty plasmid serve as positive and negative control, respectively. Oxacillin susceptibility assay was performed in quadruplicate. The Wilcoxon-Mann-Whitney test was used to assess the significance of the difference between growth with and without oxacillin. p-value adjustment was performed using the Benjamini-Hochberg procedure; ∗ = p < 0.05 (non-parametric, unpaired). (C) Oxacillin susceptibility test on solid medium reveals that 12 nt seem to be the minimal SRL for functionality of synthetic sRNAs. A representative replicate is shown. (D) Oxacillin susceptibility assay in liquid culture shows similar results. However, in liquid media an SRL of 11 nt already shows regulation, presumably based on the increased forces onto the bacterial cell wall compared with growth on solid substrate. Binding energies were calculated by IntaRNA (for display reasons the values are multiplied by −1). Oxacillin susceptibility assay was performed in quadruplicate. The Wilcoxon-Mann-Whitney test was used to assess the significance of the difference between growth with and without oxacillin. p-value adjustment was performed using the Benjamini-Hochberg procedure; ∗ = p < 0.05 (non-parametric, unpaired). pP_L-RybB-s8 is abbreviated as s8.

Figure 3

Hfq is required for functionality of synthetic RybB sRNAs (A) Liquid growth oxacillin susceptibility assay for selected RybB candidates in Δhfq. No regulation of the acrA target can be observed, which is consistent with the solid media susceptibility assay (Figure S5). The analysis indicates a reduced viability of Δhfq in contrast to the wild type (wt) and ΔacrA, already in the absence of oxacillin. Oxacillin susceptibility assay was performed in quadruplicate. The Wilcoxon-Mann-Whitney test was used to assess the significance of the difference between growth with and without oxacillin. p-value adjustment was performed using the Benjamini-Hochberg procedure; ∗ = p < 0.13 (non-parametric, unpaired). (B) The functionality of selected RybB candidates is validated by a previously established sYFP2-reporter assay using the chromosomal acrA-9′-syfp2 construct. The sRNAs show functionality in the presence of Hfq. In the absence of Hfq a fluorescence reduction of 50% can be observed for all sRNAs irrespective of the SRL. The control (ctrl) refers to empty plasmid pSL009. A wild type (wt) and Δhfq strain without reporter construct were used as background controls, respectively. Bars represent the mean of three biological replicates and error bars indicate the standard deviation. (C) Comparison of abundance and processing of selected RybB sRNAs in wild type (wt) and Δhfq by northern blot analysis. The control (ctrl) refers to empty plasmid pSL009. 5S rRNA serves as a loading control. Abundance of RybB sRNAs is reduced on average to 37% +/− 16% (RI = relative intensity in Δhfq in comparison to wild type).

Figure 4

Investigation of RNase-mediated processing of synthetic RybB sRNAs (A) Northern blot analysis of synthetic RybB sRNAs in an RNase III deletion strain (rnc⁻) and an isogenic wild type (rnc⁺). The control (ctrl) refers to empty plasmid pSL009. 5S rRNA serves as a loading control. (B) Northern blot analysis of synthetic RybB sRNAs in a temperature-sensitive RNase E background (rne^ts) and an isogenic wild type (rne⁺). The control (ctrl) refers to empty plasmid pSL009. 5S rRNA serves as a loading control. (C) Distinct processing patterns with stable intermediates can be observed which matches potential RNase E cleavage sites. SRL26, SRL52, SRL64, and SRL72 are visualized because of the first appearance of the processing band in the northern blot of the whole SRL library (see Figure S2). Theoretical and detected processing pattern exemplary visualized for RybB SRL82. For the full northern blot panel of RybB SRL library see Figure S2.

Figure 5

Adaptation of the SRL-library approach toward the SgrS sRNA for acrA targeting (A) Predicted structure of SgrS according to RNAcentral visualizes the complex 5′ secondary structure and the 3′ terminator stem-loop. For simplification a schematic is introduced for the subsequent figures. The seed region is indicated in purple and was adapted from Figure S2 of Na et al. 2013. (B) Schematic of the Golden Gate assembly of the SRL-library using SgrS as scaffold. In contrast to RybB, the promoter sequence and the 5′ sequence of SgrS are fused allowing reuse of the PCR-amplified SRL-library. All other steps are identical to the cloning of RybB (Figure 1B). (C) Screening of the SgrS candidate library. 380 candidates and four respective controls were spotted from fresh overnight cultures into 3 × 3 grids on LB-agar plates with and without oxacillin. In contrast to the RybB candidate library, fewer candidates show reduced growth under the screening condition. Violet box indicates the position of indicated controls. (D) Liquid growth analysis indicates regulation of acrA for some of the selected candidates by accessing the area under the curve (AUC) in the presence and absence of oxacillin. According to the assay at least 14 nt are needed for regulation of acrA. Synthetic SgrS sRNAs with an SRL of 36 and 42 nt are the best performing. The SRL does not correlate with its functionality despite possessing lower binding energies. Binding energies are calculated by IntaRNA (for display reasons the values are multiplied by −1). Wild type (wt) and ΔacrA with an empty plasmid are serving as respective positive and negative controls. Oxacillin susceptibility assay was performed in quadruplicate. The Wilcoxon-Mann-Whitney test was used to assess the significance of the difference between growth with and without oxacillin after p-value adjustment using the Benjamini-Hochberg procedure; ∗ = p < 0.05 (non-parametric, unpaired). (E) The minimum SRL for functionality of synthetic SgrS sRNAs was determined by oxacillin susceptibility assays on solid media. Similar to RybB, an SRL of 12 nt seems to be sufficient for regulation. A representative replicate is shown.

Figure 6

Synthetic SgrS sRNA functionality is affected by 1-nt changes (A) Solid growth oxacillin susceptibility assay for SgrS SRL36 (upper panels) and SRL42 (lower panels) in comparison to synthetic SgrS sRNAs targeting acrA mRNA that deviate by 1 nt in their SRL. A representative replicate is shown. (B) Susceptibility test for the same strains as in (A) under liquid growth conditions. Oxacillin susceptibility assay was performed in quadruplicate. The Wilcoxon-Mann-Whitney test was used to assess the significance of the difference between growth with and without oxacillin. p-value adjustment was performed using the Benjamini-Hochberg procedure; ∗ = p < 0.05 (non-parametric, unpaired).

Figure 7

Regulation of acrA by selected synthetic SgrS sRNAs in wild type and Δhfq (A) Fluorescence of reporter strain acrA-9′-syfp2 is normalized to the control strain, containing empty plasmid pSL009. In the wild type, reduction of fluorescence indicates sRNA functionality and correlates with phenotypes (cf.Figure 5D). In the absence of Hfq, the fluorescence intensity is generally increased. However, the Hfq dependence decreases with increasing SRL, and regulation by SgrS SRL82 is almost comparable between wild type and Δhfq. The control (ctrl) refers to empty plasmid pSL009. A wild type (wt) and Δhfq strain without reporter construct were used as background controls, respectively. Bars represent the mean of three biological replicates and error bars indicate the standard deviation. (B) The control (ctrl) refers to empty plasmid pSL009. 5S rRNA serves as a loading control. Abundance of SgrS sRNAs is only slightly reduced on average to 83% +/− 44% (RI = relative intensity in Δhfq in comparison to wild type).